【作者】 杨英；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2007， 博士

【摘要】 甘草被尊称为“国老”,在中医中常有“十方九草”之说,具有补脾益气、清热解毒、止咳化痰、缓急止痛、调和诸药之功效。解决甘草资源短缺的最有效的方法之一是通过细胞培养的方式来生产有价值的活性物质。本文旨在建立甘草细胞培养高产合成甘草黄酮的方法,主要围绕甘草细胞系的建立、甘草黄酮快速检测方法的建立以及提高甘草黄酮产量的方法等方面开展了较为系统的研究,取得了以下的成果:(1)筛选得到高产黄酮不产甘草酸的甘草细胞系,并对高产黄酮细胞系进行了特征分析。结果表明,用浓硫酸处理40min后,甘草种子的萌发率最高。在甘草愈伤组织诱导形成时,采用MS+2,4-D (1.0 mg L-1)+6-BA (1.0 mg L-1)+NAA (1.0 mg L-1)培养基并以下胚轴为外植体的诱导率最高,达到90%,最佳继代培养基为MS+NAA (0.5 mg L-1)+2,4-D (0.5 mg L-1)+6-BA (0.5 mg L-1)。胀果甘草细胞合成甘草总黄酮含量最高,其在固体培养基上的生长曲线呈“S”型,细胞在快速生长末期第26天左右继代比较合适,能够保持细胞良好的生长状态。(2)分析了悬浮培养的甘草细胞中甘草黄酮的成分,并建立了快速检测甘草黄酮的方法。薄层层析结果表明,甘草根中的主要黄酮成分与甘草细胞中的黄酮成分基本相同。通过液质联用方法,确定细胞中含有的黄酮物质相对分子质量为593、559、270、309、487、589、309。此外,黄酮类化合物特有的显色反应表明,甘草细胞中的黄酮类是不包含邻二酚羟基的黄酮、黄酮醇、二氢黄酮类。(3)建立了稳定的甘草细胞悬浮培养体系,确定了甘草细胞悬浮培养的最佳培养基。其中,选择MS基本培养基,接种量为4%-6%,在一个培养周期内,悬浮培养的甘草细胞的生长曲线呈“S”型,第21天干重、鲜重和黄酮总产量都达到了最高值,分别为16.4 g L-1、232.4g L-1和95.7 mg L-1。(4)研究了在逐级放大摇瓶中悬浮培养甘草细胞的生长和黄酮合成的规律。随着摇瓶体积的增加,细胞的最大生物量、黄酮产量以及最大比生长速率都在不同程度的下降,营养消耗也存在一定的差异。在逐级放大的摇瓶中,氮、磷浓度都随着培养时间延长而逐渐降低,在细胞生长对数期,三种摇瓶中磷基本都被消耗完了。对于碳源,蔗糖在培养基中转化为能够被植物细胞直接利用的还原糖,并且在培养的第22天几乎被利用完了。因此在反应器中扩大培养时,必须要充分考虑溶解氧的问题,进一步优化培养条件。(5)研究了不同浓度的水杨酸、茉莉酸甲酯和真菌多糖对甘草悬浮培养细胞合成黄酮的诱导作用。结果表明,一定浓度的茉莉酸甲酯、水杨酸和真菌多糖能够诱导提高甘草总黄酮的产量,而且导致细胞中H2O2含量升高,苯丙氨酸裂解酶、过氧化氢酶、过氧化物酶活性的增强和丙二醛含量升高。这说明一定浓度的茉莉酸甲酯、水杨酸和真菌多糖能够引起细胞产生防御反应,同时促进甘草黄酮的积累。(6)研究了前体物质苯丙氨酸、酪氨酸、肉桂酸和乙酸钠的添加对悬浮培养甘草细胞生长和甘草黄酮合成的影响。结果表明,4种前体在合适的浓度下对细胞的生长没有抑制作用,而且均能促进甘草黄酮的生物合成。苯丙氨酸的最佳添加浓度为20 mg L-1,酪氨酸、肉桂酸和乙酸钠的最佳添加浓度都是5 mg L-1,此时,均可使培养体系的黄酮产量高达100 mg L-1以上,其中酪氨酸的添加使得黄酮产量为对照的1.43倍。苯丙氨酸、肉桂酸和乙酸钠3种前体的添加时间均以第10天为宜,酪氨酸添加时间以第5天为最佳。前体物质的混合添加对黄酮合成的影响高于单独添加,这说明不同的前体之间可能存在协同作用。(7)研究了干旱胁迫对甘草细胞的生长及黄酮积累的影响,以考察甘草细胞的耐旱特征及其与甘草黄酮合成的关系。结果表明,适当的干旱胁迫能够促进甘草黄酮的积累。干旱胁迫导致甘草细胞产生大量的H2O2,而且提高了过氧化氢酶和过氧化物酶活性,提高了丙二醛的含量。这说明适当的干旱胁迫能够增强细胞防御体系的抗氧化性酶活,以增强细胞的抗旱性,同时促进甘草黄酮的积累。(8)研究了不同种类的渗透压调节剂对甘草细胞生长和黄酮合成的影响。结果表明,合适浓度的甘露醇、KCl、蔗糖的添加,在一定程度上均能促进悬浮培养的甘草细胞合成甘草黄酮。在培养过程中,KCl、甘露醇通过调节渗透压的方式促进黄酮的合成。而蔗糖,一方面,它可以作为碳源,水解为果糖和葡萄糖,供细胞直接利用;另一方面,它可能还参与调节渗透压,最终促进黄酮的合成。更多还原

【Abstract】 Licorice has been used as a traditional Chinese medicine extensively for over 2000 years. It not only has antiinflammatory, antibacterial and antiviral activities, but also has immunomodulating, antioxidant and free radical scavenging activities. Glycyrrhiza inflata Batal, an important specie of licorice, has been frequently used to treat phthisis, contagious hepatitis, ague and gastric diseases. One of the major compounds, flavonoids has been demonstrated to carry significant biological or anti-oxidation activities. Thus, the plant of G. inflata has fallen short of supply with an increasing demand for flavonoids. Plant cell culture is a useful method for the production of valuable secondary metabolites. The cell growth and the flavonoids biosynthesis are sensitive to intracellular and environmental conditions. Thus, the present study mainly focused on the establishment of cell suspension culture and of the method for flavonoids determination, and the study of regulation of flavonoids accumulation. The main results were shown as follows:(1) The culture conditions of Glycyrrhiza inflata Bat. were studied, and the cell was obtained with high flavonoids and no glycyrrhizic acid detected. The results showed that the germinations of G. inflata were the highest when treated by H2SO4 with 40min. When the hypocotyl and cotyledon were cultured on MS medium with added different kinds of hormones, the best medium to induce callus was MS+2,4-D (1.0 mg L-1)+6-BA (1.0 mg L-1)+NAA (1.0 mg L-1) with 90% induction rate. Similarly, the callus was cultured well in the medium of MS+NAA (0.5 mg L-1)+2,4-D (0.5 mg L-1)+6-BA (0.5 mg L-1). Biomass accumulation of G. inflata exhibited“S”curve in one culture cycle when the cells were cultured with solid medium, with the greatest values obtained on day 26.(2) The component of the flavonoids in cell suspension culture was analyzed, and a method for flavonoids determination was established. The results showed that the most component of the flavonoids in plant almost detected in cell suspension culture. The molecular mass of 593, 559, 270, 309, 487, 589 and 309 were ensured in the component of the flavonoids by LC-MS. Moreover, characteristic reaction indicated that the flavonoids were composed of flavone, flavonol and flavonone.(3) The stable cell suspension culture was established. When the cells were cultured in the optimized MS medium with the 4%-6% inoculum, the biomass of the cells and the flavonoids production were higher. In one culture cycle,the biomass accumulation exhibited“S”curve, with the greatest values obtained on day 21 (16.4 and 232.4 g L-1 based on dry mass and fresh mass, respectively). Similarly, flavonoids production also got to a peak of 95.7 mg L-1 on day 21.(4) The cell biomass, flavonoids production and nutrient consumption including nitrate, ammonium and phosphate during cultivation were investigated in progressive scale-up shake flasks to understand the characteristic of cell suspension culture of G. inflata during the scaling up process. The maximum biomass, flavonoids production and the maximum specific growth rate all decreased with progressive scale-up flasks. With regard to nutrient consumption, the trends of phosphate, nitrate and ammonium consumption of cells grown in progressive scale-up flasks were very similar. The phosphate concentration in the three levels of flasks was almost exhausted at the logarithm phase. Additionally, in any one shake flask, nitrate and ammonium both decreased. With regard to the carbon source, sucrose was hydrolyzed to glucose and fructose directly absorbed by cells, which was exhausted on day 22. Consequently, a conclusion could be drawn that the maximum biomass and flavonoids production decreased to some extent with the progressive scale-up flasks, and the main culture conditions such as the KLa needed to be taken into account and further optimized during the further development of G. inflata culture process for efficient and higher flavonoids production on a bioreactor scale.(5) The effects of different concentration of methyl jasmonate, salicylic acid and fungal polysaccharide on the cell biomass and flavonoids production in cell suspension cultures of G. inflata were studied. The results showed that the addition of appropriate concentration of the three kinds of elicitors resulted in the defense response,which increased the expression of the antioxidant system such as catalases and peroxidases, and enhanced the flavonoids accumulation finally.(6) The effect of four precursors on flavonoids biosynthesis in cell suspension cultures of G. inflata has been studied. The results showed that all these precursors could promote the flavonoids biosynthesis. Phenylalanine, tyrosine, cinnamic acid and NaAc have the most significant promoting effect on flavonoids synthesis at the concentration of 20, 5, 5 and 5 mg L-1, respectively. Moreover, the optimum time for tyrosine addition is the 5th day during the cell culture, while the optimum time for the other three kinds of precursors is the 10th day during the cell culture. Of the four precursors, tyrosine is the most effective. The maximum flavonoids production reached 113.04 mg L-1, 1.43 times as that of the control. The addition of the two kinds of precursors on the cell suspension culture promoted the flavonoids accumulation more, which suggested that the interaction effect exist.(7) The effect of water deficit on flavonoid production and physiological parameters for oxidative stress were studied in cell suspension culture of G. inflata to investigate its drought tolerance. The result indicated that appropriate water deficit enhanced flavonoid production. Drought stress led to hydrogen peroxide accumulation more than the control. Moreover, under drought conditions, malondialdehyde content, the activities of catalase and peroxidase increased to a greater extent than the control. All above suggested that appropriate water deficit could activate the antioxidative defense enzymes system to maintain stability in plants subjected to drought stress.(8) The effects of different kinds of osmotic regulator on the cell growth and flavonoids accumulation were studied. The results showed that the appropriate concentration of mannitol, KCl and sucrose addition improved the flavonoids production. During the cultivation, both mannitol and KCl could regulate the osmotic to promote the flavonoids accumulation. As to sucrose, it not only could be hydrolyzed to the fructose and glucose used directly by cell, but also might be considered as the osmotic regulator to improve the flavonoids production.更多还原